Our overall goal is to develop highly functional hepatocellular tissue constructs that can be used in the treatment of liver failure. While there has been reasonable progress towards development of cell-based therapies for liver disease, the key determinants of high levels of liver- specific function in isolated hepatocytes have remained elusive. We and others have shown that co-cultivation of hepatocytes with non- parenchymal cells increases hepatocellular function in vitro; however, the fundamental modulators of hepatocellular function have yet to be clearly elucidated. The limitations in our understanding of relevant hepatocellular cues is, in part, due to the spatial complexity of multicellular constructs. We have shown previously that micropatterning (spatial localization of cells on solid substrates in patterns) can be utilized to control and study key cell-cell interactions. It is our hypothesis that the same cellular constituents in different spatial configurations will produce variations in the function of the resultant tissue. In this proposal, therefore, we aim to define the relationship between tissue architecture and tissue function in co-cultures of hepatocytes and non-parenchymal cells. A clear picture of the interplay between homotypic (hepatocyte/hepatocyte) interaction, heterotypic (hepatocyte/non- parenchymal cell) signaling, and cell-matrix interactions will be fundamental to the future design and implementation of hepatocyte-based tissue engineered medical products. Towards this end, our specific aims are: l). To determine the role of homotypic (hepatocyte/hepatocyte) interactions on hepatocellular function. Homotypic interactions will be varied (from single hepatocytes to multicellular islands), and cultures probed for markers of liver-specific function. The role of ECM cues and gap junctions- normally found at the homotypic interface- will also be examined in this context. 2). To investigate the role of heterotypic (hepatocyte/non-parenchymal cell) interaction on hepatocellular function. Co-cultures of rat hepatocytes and 3T3 fibroblasts will be micropatterned such that the spatial arrangement of both cell types is specified. We have previously shown that spatial configuration may influence hepatocyte function through either: amount of heterotypic interaction or shape of hepatocellular islands. These key parameters of tissue micro-architecture will be varied and co-cultures probed for markers of liver-specific function to isolate the determinants of tissue function. The role of ECM cues and heterotypic gap junctions-putative mediators of the co-culture 'effect' will be examined. 3).To optimize functionality of tissue constructs for tissue engineering applications. We will investigate the functional consequences of reduction in non-parenchymal number as well increased heterotypic interaction through use of 3-dimensional, grooved, micropatterned substrates. These studies will enable the formation of a fundamental framework that will guide the design and implementation of current and future hepatocyte-based medical therapies.